Yeast Two-Hybrid Assay to Identify Interacting Proteins

2018 ◽  
Vol 95 (1) ◽  
pp. e70 ◽  
Author(s):  
Aurora Paiano ◽  
Azzurra Margiotta ◽  
Maria De Luca ◽  
Cecilia Bucci
Keyword(s):  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Two Hybrid

Identification of Interacting Proteins Using the Yeast Two-Hybrid Screen

2006 ◽  
pp. 211-232
Author(s):  
Kelly L. Jordan-Sciutto ◽  
Marshall B. Montgomery
Keyword(s):  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Two Hybrid

scholarly journals Evaluation of Interactions of Human Cytomegalovirus Immediate-Early IE2 Regulatory Protein with Small Ubiquitin-Like Modifiers and Their Conjugation Enzyme Ubc9

2001 ◽  
Vol 75 (8) ◽  
pp. 3859-3872 ◽  
Author(s):  
Jin-Hyun Ahn ◽  
Yixun Xu ◽  
Won-Jong Jang ◽  
Michael J. Matunis ◽  
Gary S. Hayward
Keyword(s):  
Hcmv Infection ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
Binding Assays ◽  
Yeast Two Hybrid ◽  
Vitro Binding ◽  
Lysine Residues ◽  
Infected Cells ◽  
Two Hybrid

ABSTRACT The human cytomegalovirus (HCMV) major immediate-early protein IE2 is a nuclear phosphoprotein that is believed to be a key regulator in both lytic and latent infections. Using yeast two-hybrid screening, small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) and a SUMO-conjugating enzyme (Ubc9) were isolated as IE2-interacting proteins. In vitro binding assays with glutathioneS-transferase (GST) fusion proteins provided evidence for direct protein-protein interaction. Mapping data showed that the C-terminal end of SUMO-1 is critical for interaction with IE2 in both yeast and in vitro binding assays. IE2 was efficiently modified by SUMO-1 or SUMO-2 in cotransfected cells and in cells infected with a recombinant adenovirus expressing HCMV IE2, although the level of modification was much lower in HCMV-infected cells. Two lysine residues at positions 175 and 180 were mapped as major alternative SUMO-1 conjugation sites in both cotransfected cells and an in vitro sumoylation assay and could be conjugated by SUMO-1 simultaneously. Although mutations of these lysine residues did not interfere with the POD (or ND10) targeting of IE2, overexpression of SUMO-1 enhanced IE2-mediated transactivation in a promoter-dependent manner in reporter assays. Interestingly, many other cellular proteins identified as IE2 interaction partners in yeast two-hybrid assays also interact with SUMO-1, suggesting that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interactions between IE2 and other SUMO-1-modified or SUMO-1-interacting proteins. When we investigated the intracellular localization of SUMO-1 in HCMV-infected cells, the pattern changed from nuclear punctate to predominantly nuclear diffuse in an IE1-dependent manner at very early times after infection, but with some SUMO-1 protein now associated with IE2 punctate domains. However, at late times after infection, SUMO-1 was predominantly detected within viral DNA replication compartments containing IE2. Taken together, these results show that HCMV infection causes the redistribution of SUMO-1 and that IE2 both physically binds to and is covalently modified by SUMO moieties, suggesting possible modulation of both the function of SUMO-1 and protein-protein interactions of IE2 during HCMV infection.

Identification of Connexin-Interacting Proteins: Application of the Yeast Two-Hybrid Screen

Methods ◽  
2000 ◽  
Vol 20 (2) ◽  
pp. 219-231 ◽  
Author(s):  
Chengshi Jin ◽  
Alan F. Lau ◽  
Kendra Dean Martyn
Keyword(s):  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Two Hybrid

scholarly journals Identifying Interacting Proteins of Arabidopsis Cyclophilin38 (AtCYP38), a Key Factor for PSII Assembly/Repair, via Multiple Screening Approaches

2020 ◽  
Author(s):  
Yaqi Hao ◽  
Jiashu Chu ◽  
Lujing Shi ◽  
Cong Ma ◽  
Liangliang Hui ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Thylakoid Membrane ◽  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Working Mechanism ◽  
Web Based ◽  
Thylakoid Lumen ◽  
Target Proteins ◽  
Thylakoid Membrane Proteins ◽  
Two Hybrid

Abstract BackgroundAtCYP38, a thylakoid lumen localized immunophilin, is essential for photosystem II (PSII) assembly and maintenance, but how AtCYP38 functions in chloroplast remains unknown. Based on previous functional studies and its crystal structure, we hypothesize that AtCYP38 should function via binding its targets or cofactors in the thylakoid lumen to influence PSII performance. Therefore, identifying its target proteins and cofactors would be a key step to understand the working mechanism of AtCYP38.ResultsTo identify potential interacting proteins of AtCYP38, we first adopted two web-based tools, ATTED-II and STRING, and found 15 proteins functionally related to AtCYP38. We then screened a yeast two-hybrid library including an Arabidopsis genome wide cDNA with the N-terminal domain, the C-terminal domain, and the full-length mature protein of AtCYP38. 25 positive targets were identified, but a very limited number of target proteins were localized in the thylakoid lumen. In order to specifically search interacting proteins of AtCYP38 in the thylakoid lumen, we created a yeast two-hybrid mini library including the thylakoid lumenal proteins and lumen fractions of thylakoid membrane proteins. After screening the mini library with 3 different forms of AtCYP38, we obtained 6 thylakoid membrane proteins and 9 thylakoid lumenal proteins as interacting proteins of AtCYP38. We further confirmed the localization of several identified proteins and their interaction between AtCYP38.ConclusionsAfter analysis with two web-based tools and yeast two-hybrid screenings against two different libraries, we identified a couple of potential interacting proteins, which could be functionally related to AtCYP38. We believe that the results will lay a foundation for unveiling the working mechanism of AtCYP38 in photosynthesis.

scholarly journals STOML2 Interacts with PHB through Activating MAPK Signaling Pathway to Promote Colorectal Cancer Proliferation.

2020 ◽  
Author(s):  
Wenhui Ma ◽  
Yuehong Chen ◽  
Wenyi Li ◽  
Zhuoluo Xu ◽  
Zhigang Wei ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Signaling Pathway ◽  
Regulatory Mechanism ◽  
Biological Process ◽  
Mapk Signaling ◽  
Mapk Signaling Pathway ◽  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Cancer Proliferation ◽  
Two Hybrid

Abstract BackgroundOverexpression of STOML2 has been widely reported in a variety of cancer, yet few has detailed its function and regulatory mechanism. This study aims to reveal the clinicopathologic significance and oncologic function of STOML2 in colorectal cancer, explore its specific mechanism by means of yeast two-hybrid assay and bioinformatics.MethodsExpression level of STOML2 in normal colon and CRC tissue from biobank in Nanfang Hospital was detected by pathologic methods. The malignant proliferation of CRC induced by STOML2 was validated via gain-of-function and loss-of-function experiments, with novel techniques applied, such as organoid culture, orthotopic model and endoscopy monitoring. Yeast two-hybrid assay was conducted to screen interacting proteins of STOML2, followed by bioinformatics to predict biological process and signaling pathway of candidate proteins. Target protein with most functional similarity to STOML2 was validated with co-immunoprecipitation, and immunofluorescence were conducted to co-localize STOML2 and PHB. Pathway regulated by STOML2 was detected with immunoblotting, and subsequent experimental therapy was conducted with RAF inhibitor Sorafenib.ResultsSTOML2 was significantly overexpressed in colorectal cancer and its elevation was associated with unfavorable prognosis. Knockdown of STOML2 suppressed proliferation of colorectal cancer, thus attenuated subcutaneous and orthotopic tumor growth, while overexpressed STOML2 promoted proliferation in cell lines and organoids. A list of 13 interacting proteins was screened out by yeast two-hybrid assay. DTYMK and PHB were identified to be most similar to STOML2 according to bioinformatics in terms of biological process and signaling pathways; however, co-immunoprecipitation confirmed interaction between STOML2 and PHB, rather than DTYMK, despite its highest rank in previous analysis. Co-localization between STOML2 and PHB was confirmed in cell lines and tissue level. Furthermore, knockdown of STOML2 downregulated phosphorylation of RAF1, MEK1/2, ERK1/2 and ELK1 on the MAPK signaling pathway, indicating common pathway activated by STOML2 and PHB in colorectal cancer proliferation.ConclusionsThis study demonstrated that in colorectal cancer, STOML2 expression is elevated and interacts with PHB through activating MAPK signaling pathway, to promote proliferation both in vitro and in vivo. In addition, combination of screening assay and bioinformatics marks great significance in methodology to explore regulatory mechanism of protein of interest.

scholarly journals Interaction of Mouse Polycomb-Group (Pc-G) Proteins Enx1 and Enx2 with Eed: Indication for Separate Pc-G Complexes

1998 ◽  
Vol 18 (6) ◽  
pp. 3572-3579 ◽  
Author(s):  
Maarten van Lohuizen ◽  
Marieke Tijms ◽  
Jan Willem Voncken ◽  
Armin Schumacher ◽  
Terry Magnuson ◽  
...  
Keyword(s):  
G Proteins ◽  
Polycomb Group ◽  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Sex Combs ◽  
Embryonic Ectoderm Development ◽  
Transient Interactions ◽  
Embryonic Ectoderm ◽  
Two Hybrid

ABSTRACT The Polycomb group (Pc-G) constitutes an important, functionally conserved group of proteins, required to stably maintain inactive homeobox genes repressed during development. Drosophila extra sex combs (esc) and its mammalian homolog embryonic ectoderm development (eed) are special Pc-G members, in that they are required early during development when Pc-G repression is initiated, a process that is still poorly understood. To get insight in the molecular function of Eed, we searched for Eed-interacting proteins, using the yeast two-hybrid method. Here we describe the specific in vivo binding of Eed to Enx1 and Enx2, two mammalian homologs of the essential DrosophilaPc-G gene Enhancer-of-zeste[E(z)]. No direct biochemical interactions were found between Eed/Enx and a previously characterized mouse Pc-G protein complex, containing several mouse Pc-G proteins includingmouse polyhomeotic (Mph1). This suggests that different Pc-G complexes with distinct functions may exist. However, partial colocalization of Enx1 and Mph1 to subnuclear domains may point to more transient interactions between these complexes, in support of a bridging role for Enx1.

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